The invention relates to biosynthetic maturation of cell surface polypeptides and, more specifically, to particular CFTR polypeptides which exhibit increased transport to the cell surface and tripeptide amino acid sequences that promote or enhance transport of export-incompetent CFTR to the cell surface.
Large multidomain and multisubunit proteins are assembled into their native tertiary and quaternary structures, respectively, in the endoplasmic reticulum (ER). If this assembly is imperfect because of mutations or for other reasons, the aberrant protein is targeted for degradation by a set of processes generally referred to as biosynthetic quality control. Although a great deal has been learned about these processes in the past few years the rate-limiting step determining whether or not a nascent chain is exported from the ER has not been identified. Indeed, the relation of the folding and degradation mechanisms to the ER export and retrieval pathways is not understood for any cell surface or secreted molecule.
Cystic fibrosis (CF) is an example of a disease which may benefit from an understanding of the factors that contribute to or mediate retention or export of proteins from the endoplasmic reticulum because, in many patients, ER-retention prevents potentially functional variant CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) molecules from reaching the plasma membranes of secreting and reabsorbing epithelial cells, where CFTR is required as a regulated chloride channel. For example, although more than 800 different mutations in the CFTR gene have been detected in CF patients, more than 90% of patients possess a single misprocessing mutant, ΔF508. Collins, F. S. (1992) Science 256:774-779. This mutant protein is potentially functional as a regulated chloride channel if it can be made to move further along the secretory pathway and reach the cell surface. Dalemans, W. et al. (1991) Nature 354:526-528; Drumm, M. L. et al. (1991). Science 254:1797-1799; and Li, C. et al. (1993) Nat. Genetics 3:311-316.
Ubiquitination ultimately marks maturation-incompetent nascent chains as substrates for the proteasome but earlier steps in the recognition of these targets are unclear. Jensen, T. J. et al. (1995) Cell 83:129-135; Sato, S. et al. (1998). J. Biol. Chem. 273:7189-92; and Ward, C. L et al. (1995) Cell 83:121-127. Molecular chaperones can either retain unfolded proteins or assist in their folding. Nascent CFTR interacts with chaperones on both sides of the ER membrane; on the cytoplasmic face of the ER, Hsp70 (Yang, Y. et al. (1993) Proc. Natl. Acad. Sci. USA 90:9480-9484) and its cochaperone, Hdj-2 (Meacham, G. C. et al. (1999) EMBO J. 18:1492-1505) as well as Hsp90 (Loo et al. (1998) EMBO J. 17:6879-6887) bind to immature CFTR. Other chaperones could also be present in the large multimolecular complexes containing nascent CFTR. Pind, S. et al. (1994) J. Biol. Chem. 269:12784-12788. Although these interactions appear to occur with both wild-type CFTR and mutant ΔF508 CFTR and no large differences in the kinetics or stoichiometry of CFTR-chaperone interactions have yet been found, the role of chaperones in ER retention of CFTR cannot be ruled out.
In addition to chaperones, short sequence motifs have been shown to exert positive and negative effects on secretory proteins which are required for ER export and retrieval, respectively. For example, a short diacidic ER export signal has been described as necessary for transport of VSV-G glycoprotein from the ER. Nishimura, N., and Balch, W. E. (1997) Science 277:556-8. Whether nascent ΔF508 CFTR never leaves the ER or if it is retrieved is not known, although ΔF508 CFTR may reach the intermediate compartment (ERGIC) between ER and Golgi. Gilbert, A. et al. (1998) Exp. Cell Res. 242:144-52.
Previous attempts to overcome ER-retention of mutant CFTR have included inhibition of the proteasome involved in nascent chain proteolysis (Jensen et al., 1995, supra; Ward et al., 1995, supra), perturbation of interaction with molecular chaperons (Jiang, C. et al. (1998). Am. J. Physiol. 275:C171-8; and Loo et al., 1998, supra) and using agents or conditions which influence protein folding for example, glycerol and other osmolytes (Brown, C. R. et al. (1996) Cell Stress Chaperones 1:117-25; Qu, B. H. et al. (1997) J. Biol. Chem. 272:15739-44; and Sato, S. et al. (1996) J. Biol. Chem. 271:635-638) or reduced temperature (Denning, G. M. et al. (1992) Nature 358:761-764). However, these treatments are minimally effective or extremely toxic to cells, precluding their application to patients.
Thus, there remains a need to understand the biological basis for ER retention, especially in respect to the retention of proteins such as CFTR that have severe physiological consequences. The present invention satisfies this need and provides related advantages as well.